The clinical implementation of molecularly targeted imaging modalities has progressed considerably in recent years. Fluorophores in the NIR range are of particular interest for in vivo optical imaging due to the significant tissue penetration of light in this range. Despite a central role in modern biology and medicine, the compounds employed in NIR fluorescence techniques have changed little in recent decades. Using molecular design concepts borrowed from related fields (e.g. medicinal chemistry and modern organic synthesis), we seek to develop new agents with improved utility for cancer-related imaging and microscopy. The long-term goal is to identify readily synthesized, stable, and bright fluorophores with optimal properties for biomedical imaging. Our current efforts in this area are split in two aims. Aim 1 - Synthetic methods to prepare heptamethine cyanine fluorophores. The heptamethine cyanine class of near-IR fluorophores are used for many applications, with extensive recent progress in the context of fluorescence-guided surgery. We have developed a new rearrangement reaction that enables the synthesis of previously inaccessible variants. Compared to existing agents, the compounds we have prepared exhibit improved optical properties and significantly greater chemical stability to biological nucleophiles. Through an extensive optimization campaign, we have developed molecules that are exceptionally resistant to aggregation following labeling on both targeting antibodies and nanoparticles. These molecules exhibit reduced liver uptake and improved in vivo signal when compared to existing agents used for clinical application. We have also shown that small changes in the polar functional groups appended to these fluorophores can have a dramatic impact on biodistribution and tumor accumulation when used without targeting motifs. Through a collaboration with surgeons at Children's National Hospital we have developed molecules that be used to visualize the bile duct and ureter, both sensitive anatomical features often injured during abdominal surgery. We are currently pursuing approaches to target fluorescence molecules to solid tumors, with a parituclar focus on hepatobillary tumors. Aim 2 - Synthesis and evaluation of novel far-red probes. We have developed a chemical strategy to assemble polycyclic pentamethine cyanines through a cross metathesis/polycylcization strategy. When compared to conventional pentamethine cyanines, the resulting compounds exhibit significantly higher fluorescence quantum yield (4X) and, additionally, recover from sodium borohydride reduction with improved efficiency. These features allow these compounds to be used for super resolution microscopy and enable excellent photon counts without recourse to complex deoxygenation buffers. We have recently prepared molecules with improved antibody and nucleic acid labeling properties. We are also extending this approach to longer wavelength dyes, where this a significant need for brighter molecules.